CN104407135A - Method and kit for detecting A type influenza virus H5 and H9 subtypes - Google Patents
Method and kit for detecting A type influenza virus H5 and H9 subtypes Download PDFInfo
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- CN104407135A CN104407135A CN201410614127.2A CN201410614127A CN104407135A CN 104407135 A CN104407135 A CN 104407135A CN 201410614127 A CN201410614127 A CN 201410614127A CN 104407135 A CN104407135 A CN 104407135A
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Abstract
The invention discloses a method and a kit for detecting A type influenza virus H5 and H9 subtypes. The kit comprises a first coating membrane and a second coating membrane, and one end of the first coating membrane is connected with one end of the second coating membrane; at least one area in the first coating membrane is coated with a first antibody with a fluorescent label and a second antibody with a fluorescent label; the second coating membrane comprises a first area, a second area and a third area which are separated, and the first area and the second area are closer to the first coating membrane compared with the third area; the first area is coated with a third antibody, and the third antibody and the first antibody can be specifically combined with a same antigen; the second area is coated with a forth antibody, and the forth antibody and the second antibody can be specifically combined with a same antigen; and the third area is coated with an antiantibody, and the antiantibody can be specifically combined with the first antibody and the second antibody. The kit and/or the method for detecting the A type influenza virus H5 and/or H9 subtypes have the advantages of high sensitivity, strong specificity, rapidness, convenience, capability of realizing objective determination and the like.
Description
Technical field
The present invention relates to field of virus detection, especially, the present invention relates to detection method and the kit thereof of influenza A H5 and/or H9 hypotype, especially, the present invention relates to a kind of kit, kit in the purposes detected in influenza A H5 and/or H9 hypotype and a kind of method detecting influenza A H5 and/or H9 hypotype.
Background technology
Influenza A and influenza A virus are common flow Influenza Virus, and influenza A virus easily morphs, and its hypotype is often called " bird flu " by people.Bird flu is the crushing epidemic disease of serious harm world aviculture, its cause of disease is easily suddenlyd change, there is multiple serotype, bring difficulty to the prevention and control of this disease, can infect the mankind after viral gene variation, metainfective symptom main manifestations is high heat, cough, runny nose, myalgia etc., most with serious pneumonia, the multiple organ failures such as the severe patient heart, kidney cause death, and according to WHO statistics, people infects the mortality ratio of H5 hypotype up to 60%.Flu-A is high to human pathogenic, once repeatedly causes worldwide being very popular.So far find in influenza A virus that the avian influenza virus subtype of energy direct infection people has: H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8, wherein H1, H5, H7, H9 hypotype is highly pathogenic.
The detection method of influenza A H5 common at present and H9 hypotype mainly contains: detection of nucleic acids and Virus Isolation.Detection of nucleic acids has RT-PCR to detect and real-time RT-PCR detection method, and detection of nucleic acids depends on the quality and quantity of sample form RNA, and operation requirements is strict, and need a large amount of instrument and equipment and professional's operation, detection time is longer, and detection sensitivity is high.Virus Isolation is usually used in identifying the hypotype of positive virus, needs specialized laboratories just can carry out.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or at least provides a kind of useful business to select.
For this reason, according to an aspect of of the present present invention, a kind of kit is provided, it comprises the first coated film and the second coated film, one end of described first coated film is connected with one end of described second coated film, at least one piece of region in described first coated film is coated with the fluorescently-labeled first antibody of band and the fluorescently-labeled second antibody of band, described second coated film comprises the first area of separation, second area and the 3rd region, described first area and described second area than described 3rd region near described first coated film, described first area is coated with the 3rd antibody, described 3rd antibody and described first antibody can specific binding same antigens, described second area is coated with the 4th antibody, described 4th antibody and described second antibody can specific binding same antigens, described 3rd region is coated with antiantibody, described antiantibody can first antibody and described second antibody described in specific binding.To being coated with fluorescence labeling first antibody and having the size in the region of fluorescence labeling second antibody not to be restricted in the first coated film, it can be whole first coated film, also can be a part for the first coated film, the first coated film be called as sample pad in one embodiment of the invention.Same, the first area of the separation in the second coated film, second area and the 3rd region size separately are not also particularly limited, as long as these three regions any two or do not have between wantonly three to connect or the region of overlap, in addition, which in first area and second area is not also restricted closer to the first coated film.In one embodiment of the invention, as shown in Figure 1, first coated film of kit and the second coated film are all rectangular, a broadside of the first coated film glues mutually with a broadside of the second coated film, the region being coated with fluorescence labeling first antibody in first coated film is for being about 2cm, the rectangle of wide about 3mm, first area in second coated film, second area and the 3rd region are all wire, first area is called as detection line 1 or T1 line, second area is called as detection line 2 or T2 line, 3rd region is called as nature controlling line or C line, the wire plane at place, three regions is all parallel to the bonding limit of two coated films, said wire first, second or the 3rd the width in region be approximately 0.5-1mm, long is the wide of place film.
This kit of the present invention utilizes the first film and the second film to be coated with fluorescently-labeled first antibody and the second antibody of energy specific binding same antigen respectively, rete is utilized to analyse after adding testing sample, second film is formed double-antibody sandwich compound, based on the first antibody detected in compound with fluorescently-labeled fluorescence intensity come whether there is antigen to be checked in judgement sample.This kit of the present invention significantly can improve the specificity of detection, shortens and detects required time.By fluorescence labeling first antibody, significantly detection sensitivity can be improved.
According to one embodiment of present invention, the other end of the second coated film in this kit on the one hand of the present invention is connected with adsorptive pads.Adsorptive pads can be strong absorbent material, can give directed forces like this make liquid sample from directed chromatography to the second coated film of the first coated film when detecting liquid sample.
According to one embodiment of present invention, described first coated film, described second coated film and described adsorptive pads are fixed on same solid-phase matrix.Solid-phase matrix is mainly has a carrying when using the film in kit of the present invention, handled easily, the type of solid-phase matrix is not particularly limited, can for reacting or not affect the inert material that antigen-antibody is combined with testing sample, such as cardboard, plastic plate etc.
According to one embodiment of present invention, described first coated film is glass fibre element film, and described second coated film is nitrocellulose filter (NC film).Glass fibre membrane is chemical inertness, not containing bonding agent, adopts 100% pyrex fiber manufacture to form, it is coated with fluorescently-labeled first antibody, be beneficial to the target antigen generation specific binding in first antibody and testing sample.And NC film itself is with the addition of surfactant to improve hydrophilic ability, and had certain buffer system, there is capillary fiber structure, moisture more more than equal cellulosic filter paper can be adsorbed, flow velocity is fast, high temperature resistant, the 3rd and the/the four antibody that profit wraps quilt thereon react with aforesaid fluorescence labeling first antibody-antigen and/or fluorescence labeling second antibody-antigen generation specific binding respectively, fluorescence excitation.
According to one embodiment of present invention, described fluorescence labeling is fluorescent microsphere mark, and described first antibody is marked with described fluorescent microsphere and is combined by covalent peptide bonds.Like this, improve the stability of label, avoid the sterically hindered impact of antibody, be conducive to improving sensitivity and specificity.In one embodiment of the invention, described fluorescent microsphere diameter is in nano-scale range, and its diameter range is 10-500nm, and be preferably 20-300nm, on it, load has fluorescent material, and being stimulates the solia particle that can inspire fluorescence by outside energy.The fluorescent material of described fluorescent microsphere load is the fluorescent nano material converted of doped with fluorescent dyes, rare-earth complex, quantum dot etc.The fluorescent material of described fluorescent microsphere load is modified with active functional group group by high molecular polymer, and described active functional group group is carboxyl, amino, hydroxyl, sulfydryl.In one embodiment of the invention, described active functional group group is specially carboxyl, and described fluorescent microsphere mark first antibody is that the fluorescent microsphere of first antibody to be marked and carboxyl modified is combined the condensate formed with covalent peptide bonds.
According to one embodiment of present invention, described first antibody is influenza A H5 subclass antibodies, described second antibody is influenza A H9 subclass antibodies, described 3rd antibody is the influenza A H5 subclass antibodies being different from described first antibody, and described 4th antibody is the influenza A H9 subclass antibodies being different from described second antibody.First and the 3rd antibody can specific binding antigen A type influenze virus H 5 subtype, and preferably, the different surfaces determinant specific binding of two kinds of antibody capables and H5 hypotype, second and the 4th antibody can specific binding antigen A type influenza virus H9 hypotype, and preferably, the different surfaces determinant specific binding of two kinds of antibody capables and H9 hypotype, is beneficial to accurately carrying out of H5 and/or H9 hypotype detection like this.In one embodiment of the invention, described first antibody and/or the 3rd antibody are any one in influenza A H5 hypotype monoclonal antibody and influenza A H5 hypotype polyclonal antibody, and described second antibody and/or the 4th antibody are any one in influenza A H9 hypotype monoclonal antibody and influenza A H9 hypotype polyclonal antibody.This kit of the present invention is based on the research to fluorescence labeling, antigen and antibody characteristic, directed covalent chemical coupling is carried out by selecting the fluorescence labeling that is applicable to and specific antibody, acquisition fluorescent-labeled antibody is analyzed, and by optimizing the immunoreactive various condition of double-antibody sandwich, prepare the above-mentioned kit that can be used in detecting influenza A H5 and/or H9 hypotype.
According to one embodiment of present invention, described antiantibody is sheep anti-mouse igg antibody, its can with first antibody and/or second antibody specific binding, namely with unnecessary band fluorescence labeling first antibody and/or be with fluorescence labeling second antibody to be combined to form immune complex, fluorescence excitation, being able to can qualitative and/or quantitative this immune complex of detection.
According to another aspect of the present invention, what provide the invention described above one side is detecting the purposes in influenza A H5 and/or H9 hypotype with the arbitrary kit in various embodiment.Above for the advantage described by kit and technical characteristic, be still applicable to the purposes of this kit, do not repeat them here.
According to another aspect of the present invention, there is provided a kind of method detecting influenza A H5 and/or H9 hypotype, described method comprises step: sample to be tested is added to the region being coated with the fluorescently-labeled first antibody of band and the fluorescently-labeled second antibody of band in the first coated film in above-mentioned arbitrary kit; Detect the fluorescence intensity in the first area in described kit, second area and the 3rd region, obtain first area fluorescence intensity, second area fluorescence intensity and the 3rd region fluorescence intensity; The first predetermined threshold is greater than based on the first ratio, judge to there is influenza A H5 hypotype in described sample to be tested, and/or be greater than the second predetermined threshold based on the second ratio, judge to there is influenza A H9 hypotype in described sample to be tested, wherein, first ratio=first area fluorescence intensity/the 3rd region fluorescence intensity, the second ratio=second area fluorescence intensity/the 3rd region fluorescence intensity.The region being coated with band fluorescence labeling first antibody and band fluorescence labeling second antibody in first coated film is the region combined with the target antigen (if yes) in sample to be tested, in one embodiment of the invention, this region is called application of sample end.In one embodiment of the invention, described predetermined threshold is 0.025, this value is by reference to utilizing known double-antibody sandwich immunoreactive conditioned disjunction commercial reagent box product, large quantitative determination normal sample was originally determined originally with the normal sample adding positive sample (containing H5 and/or H9), it is an average for the ratio of the two measured value, utilize this average as critical value (cutoff), accurately can judge that detecting sample is positive or negative.In one embodiment of the invention, by hand-held instrument fluorescence intensity, obtain first area fluorescence intensity and second area fluorescence intensity respectively with the ratio of the 3rd region fluorescence intensity, by two ratios respectively compared with described critical value, objectively can obtain testing result, the testing result of the fluorescent PCR kit of this testing result and commercially available detection influenza A H5 and/or H9 hypotype is consistent.Utilize this detection method of the present invention, utilize mentioned reagent box of the present invention, can realize to influenza A H5 and/or H9 hypotype fast and Sensitive Determination, method of the present invention has highly sensitive, high specificity, quick, easy, can realize the advantage measured that objectifies.Utilize this kit of the present invention and/or method to detect influenza A H5 and/or H9 hypotype, 0.016HA Unit is reached to the sensitivity of influenza A H5 hypotype, 0.5HA Unit is reached to the sensitivity of influenza A H9 hypotype.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 is the reagent cartridge configuration schematic diagram in one embodiment of the present of invention;
Fig. 2 is the structural representation of the kit of detection influenza A H5 in one embodiment of the present of invention and H9 hypotype;
Fig. 3 is detected value and the concentration standard curve figure of detection influenza A H5 in one embodiment of the present of invention and H9 hypotype.
Embodiment
According to the embodiment of the present invention, provide following embodiment and be convenient to understand the present invention better, but do not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.In addition, the term that the term " bag quilt " in the present invention is immune field, comprises absorption, fixing meaning.
First antibody is called influenza A H5 hypotype labelled antibody below, second antibody is called influenza A H9 hypotype labelled antibody, fluorescently-labeled first antibody is called influenza A H5 subclass antibodies potpourri, fluorescently-labeled second antibody is called influenza A H9 subclass antibodies potpourri, 3rd antibody is called influenza A H5 hypotype coated antibody, 4th antibody is called influenza A H9 hypotype coated antibody, first area is called test section 1 or T1 line, second area is called test section 2 or T2 line, 3rd region is called quality control region or C line, first coated film is called sample pad, second coated film is called coated film.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Fluorescent microsphere used in following embodiment is purchased from Bangs Laboratories, Inc. company, and catalog number is FC02F/10930 solid content is 10mg/ml, and be the fluorescent microsphere of carboxyl modified, this fluorescent microsphere mean diameter is 110nm.
Influenza A H5 to be marked used in following embodiment and H9 subclass antibodies are the monoclonal antibodies being numbered F5-25, F9-17 anti-influenza type A virus H5 and H9 hypotype purchased from the large monoclonal antibody center of Kunming cloud.
Influenza A H5 in following embodiment and H9 hypotype coated antibody are the monoclonal antibodies being numbered F5-39, F9-47 anti-influenza type A virus H5 and H9 hypotype purchased from the large monoclonal antibody center of Kunming cloud.
Glass fibre membrane for making sample pad in following embodiment is GF-CP20300 purchased from Millipore company, cat. no.
Thieving paper for making adsorptive pads in following embodiment is CF-SP22300 purchased from Millipore company, cat. no.
Nitrocellulose filter for making coated film in following embodiment is Hi-Flow PlusHF135 purchased from Millipore company, cat. no.
PH in following embodiment be 7.4 0.02M PBS damping fluid prepare as follows: take 2.3g Na
2hPO
4, 0.524gNaH
2pO
4.H
2o, 8.77g NaCl is dissolved in pure water, is settled to 1L with pure water, adjusts pH to 7.4, obtains the 0.02M PBS damping fluid that pH is 7.4.
Film process damping fluid in following embodiment is prepared as follows: Tween-20, BSA, sucrose are dissolved in above-mentioned pH be 7.4 0.02M PBS damping fluid make that the mass percentage of Tween-20 is 0.2%, the mass percentage of BSA is 1%, the mass percentage of sucrose is 2%, adjust pH to 7.4, obtain film process damping fluid.
50mM pH in following embodiment be 8.5 borate buffer solution prepare as follows: take 1.9g Na
2b
4o
7.10H
2o is dissolved in 100ml pure water, adjusts pH to 8.5 to obtain the borate buffer solution that 50mM pH is 8.5.
Sample treatment liquid in following embodiment is prepared as follows: Tween-20 is dissolved in above-mentioned pH be 7.4 0.02M PBS damping fluid make the mass percentage of Tween-20 be 0.2%, adjust pH to 7.4, obtain sample treatment liquid.
The general preparation method detecting influenza A H5 and H9 hypotype kit comprises the following steps:
(1) preparation of the fluorescent microsphere label probe of carboxyl modified
Adopt the fluorescent microsphere of the carboxyl modified be applicable to, after activating the carboxyl on its surface, adopt the mode of covalent coupling respectively influenza A H5 and H9 hypotype labelled antibody orientation to be connected to the fluorescent microsphere surface of this carboxyl modified.
(2) the bag quilt of test section T line and C line place antibody
Adopt spray film instrument, in the T1 line place spraying influenza A H5 hypotype coated antibody of coated film test section, in the T2 line place spraying influenza A H9 hypotype coated antibody of coated film test section, in C line place spraying sheep anti-mouse igg antibody.
(3) the bag quilt of sample pad place label probe
Adopt spraying apparatus, in anti-influenza type A virus H5 and the H9 subclass antibodies potpourri of sample pad specific location spraying fluorescent microsphere mark.This ad-hoc location is one piece of region in sample pad, and this region is namely as follow-up " application of sample end ".
(4) assembled formation of kit
Be intended to Fig. 2 according to the structural diagrams of kit, paste the coated film as test section in the middle of plastic support backboard, T1 or the T2 line end in coated film pastes sample pad, and C line end pastes adsorptive pads.Adopt test paper cutting machine, its point is cut to the paper slip in certain broadband, and loads intermediate plate, pack with the aluminium foil bag that drying agent is housed.
(5) formation of antigen-antibody fluorescent immune complex
Testing sample is added in the application of sample end place of the reaction plate of above-mentioned assembled formation, the influenza A H5 hypotype coated antibody that after the influenza A H5 hypotype labelled antibody that influenza A H5 hypotype in sample and fluorescent microsphere mark is combined, chromatography sprays to T1 line place, form coated antibody-antigen-fluorescent microsphere labelled antibody immune complex at T1 line place, unnecessary fluorescent microsphere mark influenza A H5 and H9 subclass antibodies are then at the fluorescent mark immunity compound that C line place and sheep anti-mouse igg are formed; The influenza A H9 hypotype coated antibody that after the influenza A H9 hypotype labelled antibody that influenza A H9 hypotype in sample and fluorescent microsphere mark is combined, chromatography sprays to T2 line place, form coated antibody-antigen-fluorescent microsphere labelled antibody immune complex at T2 line place, unnecessary fluorescent microsphere mark influenza A H5 and H9 subclass antibodies are then at the fluorescent mark immunity compound that C line place and sheep anti-mouse igg are formed.
(6) fluorescent mark immunity complex fluorescence intensity detection
Measure T1 line place and T2 line place fluorescence intensity with fluorescence detector, determine its positive or negative result by the threshold value comparison with setting, C line measurement result is then marked as in the Quality Control of this assay method.
The fluorescence intensity of described fluorescent mark immunity compound, refers to the numerical value obtained after the quantity fluorescence detector of the combined with fluorescent microballoon under T1 line, T2 line and C line place are detained respectively measures.By the immunoreactive condition of double-antibody sandwich, originally can determine the mensuration average of variant normal sample with interpolation positive sample through large quantitative determination normal sample, determine in this, as critical value (cutoff) the positive or negative result detecting sample.C line measurement result is then marked as in the Quality Control of this assay method.
Embodiments of the invention are described below in detail.Embodiment is exemplary below, only for explaining the present invention, and can not be interpreted as limitation of the present invention.It should be noted that, term " first " used in this article, " second ", " the 3rd " and " the 4th " etc. only for conveniently describing object, and can not be interpreted as instruction or hint relative importance, have sequencing relation between can not being interpreted as.In describing the invention, except as otherwise noted, the implication of " multiple " is two or more.
Embodiment 1 detects the preparation of influenza A H5 and H9 hypotype kit
(1) preparation of fluorescent microsphere mark influenza A H5 and H9 hypotype labelled antibody
With mean diameter be 110nm, carboxyl modified fluorescent microsphere (purchased from Bangs Laboratories, Inc. company, catalog number is FC02F/10930), the monoclonal antibody of anti-influenza type A virus H5 and H9 hypotype (purchased from the large monoclonal antibody center of Kunming cloud be numbered F5-25, F9-17), prepare fluorescent microsphere mark influenza A H5 and H9 hypotype labelled antibody by the following method:
Get the above-mentioned carboxyl modified of 10mg fluorescent microsphere MES damping fluid (0.1M, pH4.7) washing and centrifugal after, resuspended with 1ml MES damping fluid (0.1M, pH4.7), add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to final concentration be 5mM, to add NHS (N-hydroxysuccinimide) to final concentration be 10mM, room temperature lucifuge, reaction obtains the fluorescent microsphere activating rear carboxyl modified half an hour.
Wash the fluorescent microsphere of carboxyl modified after this activation with the borate buffer solution of 50mM pH8.5, after getting the above-mentioned influenza A H5 to be marked of 0.37mg and H9 subclass antibodies and the above-mentioned activation of 5mg respectively, the fluorescent microsphere of carboxyl modified is mixed in the borate buffer solution of 50mM pH8.5 and fully mixes.React 2 hours under room temperature lucifuge, allow antibody and fluorescent microsphere form stable covalent peptide bonds and combine the conjugate obtaining fluorescent microsphere and influenza A H5 and H9 subclass antibodies.After reaction terminates, adding final concentration is that the BSA solution of 1% (mass percentage) is closed residual activity carboxyl site on the conjugate of fluorescent microsphere and influenza A H5 and H9 subclass antibodies, and room temperature lucifuge reacts 0.5 hour.After completing, mark influenza A H5 and H9 subclass antibodies liquid with the 0.02M PBS buffer solution of pH7.4, the resuspended 5mg/ml fluorescent microsphere that obtains, 4 DEG C of preservations are stand-by.
(2) preparation of influenza A H5 and H9 hypotype kit is detected
With influenza A H5 and H9 hypotype coated antibody, prepare coated film with sheep anti-mouse igg antibody, concrete grammar is as follows:
Adopt the 0.02M PBS damping fluid of pH7.4, by sheep anti-mouse igg antibody (Bo You bio tech ltd, Changsha, ABGAM-0500) concentration 1mg/ml solution is formulated as, influenza A H5 and H9 hypotype coated antibody (are numbered F5-39 purchased from the large monoclonal antibody center of Kunming cloud, F9-47) concentration is formulated as concentration 2mg/ml solution respectively, the XYZ3050 of BioDot spray membranous system is selected sheep anti-mouse igg antibody to be sprayed onto nature controlling line (C line) position of coated film (nitrocellulose filter), influenza A H5 hypotype coated antibody is sprayed onto detection line (T1 line) position, influenza A H9 hypotype coated antibody is sprayed onto detection line (T2 line) position, dehumidifier dried for standby after 4 hours is carried out in the drying plant that relative humidity is less than 10%, obtain the coated film with detection line and nature controlling line.
All-glass paper half an hour is soaked with above-mentioned film process damping fluid, the temperature of soaking is 37 DEG C, dehumidifier is carried out after 4 hours in same dehumidifier condition, after being 0.05mg/ml mixed liquor with above-mentioned film process damping fluid dilution step (one) gained fluorescent microsphere mark influenza A H5 and H9 subclass antibodies liquid to fluorescent microsphere mark influenza A H5 and H9 subclass antibodies content, adopt BioDot XYZ3050 spray membranous system be sprayed into above-mentioned process glass fibre element film on preparation formed sample pad, carry out drying in same dehumidifier condition.In 100,000 grades of cleanings and dry workshop the above-mentioned dried coated film with detection line and nature controlling line, above-mentioned sample pad, adsorptive pads, backboard by carrying out after collocation assemble shown in Fig. 2, the Paperboard cutting posted is the width of 4mm/ bar by the CM4000 cutting system of employing BioDot, loads detection intermediate plate stand-by.
Embodiment 2 detects the assessment of influenza A H5 and/or H9 hypotype kit
(1) detection sensitivity
Test antigen using influenza A H5 and H9 hypotype HI and measure the detection influenza A H5 of embodiment 1 and the sensitivity of H9 hypotype kit as testing sample.
Influenza A H5 and H9 hypotype HI are tested simultaneously antigen pH be 7.4 0.02M PBS damping fluid respectively and be hybridly prepared into series concentration (64,32,16,8,4,2,1,0.5,0.25,0.125,0.063,0.031,0.016,0.008,0HA Unit), add respectively in the application of sample end of detection influenza A H5 and the H9 hypotype kit obtained by embodiment 1, and adopt fluorescence detector fluorescence intensity.Detecting step: first detected sample is recovered room temperature (25 DEG C) before detection, get with accurate pipettor the application of sample end that detected sample 60 μ l vertically slowly instills the detection influenza A H5 that obtains of embodiment 1 and H9 hypotype kit, test with fluorescence detector after 10 minutes.
Its testing result is as shown in table 1 below.The sensitivity that can draw the detection influenza A H5 hypotype of embodiment 1 from testing result is 0.016HA Unit, and the sensitivity detecting influenza A H9 hypotype is 0.5HA Unit (T/C Cut-Off value is greater than 0.025 for positive value).
Influenza A H5 and H9 hypotype kit detected value and concentration curve are as shown in Figure 3.
The kit detected value of table 1 influenza A H5 sample concentration different from H9 hypotype
(2) accuracy detects
Choose H5 and the H9 hypotype mixing sample of 3 parts of variable concentrations, distinguish duplicate measurements according to the method described in the present invention 10 times, calculate batch interior mean deviation CV% value according to the results of 10 times.The 3 batches of kits prepared according to the method described in the present invention, choose the sample of 3 parts of variable concentrations, respectively duplicate measurements 10 times, mean deviation CV% value between criticizing according to result calculating.Its testing result is as shown in table 2 below.
In table 2 batch, difference between batch measures
(3) cross reaction detects
Other hypotypes of influenza A and other common Respirovirus HI are tested antigen, be concentration 64HA Unit with the 0.02M PBS buffer that pH is 7.4, get with accurate pipettor the application of sample end that 60 μ l vertically slowly instill the detection influenza A H5 that obtains of embodiment 1 and H9 hypotype kit respectively, test with fluorescence detector after 10 minutes.Its testing result is as shown in table 3 below.Influenza A H5 of the present invention and H9 hypotype kit and there is no cross reaction between other hypotypes of influenza A and other common Respiroviruses.
Table 3 influenza A H5 and the cross reaction of H9 hypotype kit detect
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For those skilled in the art, without departing from the inventive concept of the premise, some equivalent to substitute or obvious modification can also be made, and performance or purposes identical, all should be considered as belonging to protection scope of the present invention.
Claims (10)
1. a kit, it comprises the first coated film and the second coated film, and one end of described first coated film is connected with one end of described second coated film,
At least one piece of region in described first coated film is coated with the fluorescently-labeled first antibody of band and the fluorescently-labeled second antibody of band,
Described second coated film comprises the first area of separation, second area and the 3rd region, described first area and described second area than described 3rd region near described first coated film,
Described first area is coated with the 3rd antibody, and described 3rd antibody and described first antibody can specific binding same antigens,
Described second area is coated with the 4th antibody, and described 4th antibody and described second antibody can specific binding same antigens,
Described 3rd region is coated with antiantibody, and described antiantibody can first antibody and described second antibody described in specific binding.
2. the kit of claim 1, is characterized in that, the other end of described second coated film is connected with adsorptive pads.
3. the kit of claim 2, is characterized in that, described first coated film, described second coated film and described adsorptive pads are fixed on same solid-phase matrix.
4. the kit of claim 1, is characterized in that, described first coated film is glass fibre element film, and described second coated film is nitrocellulose filter.
5. the kit of claim 1, is characterized in that, described fluorescence labeling is fluorescent microsphere mark.
6. the kit of claim 5, is characterized in that, described first antibody is marked with described fluorescent microsphere and is combined by covalent peptide bonds, and described second antibody is marked with described fluorescent microsphere and is combined by covalent peptide bonds.
7. the kit of claim 1, it is characterized in that, described first antibody is influenza A H5 subclass antibodies, described second antibody is influenza A H9 subclass antibodies, described 3rd antibody is the influenza A H5 subclass antibodies being different from described first antibody, and described 4th antibody is the influenza A H9 subclass antibodies being different from described second antibody.
8. the kit of claim 1, is characterized in that, described antiantibody is sheep anti-mouse igg antibody.
9. the arbitrary kit of claim 1-8 is detecting the purposes in influenza A H5 and/or H9 hypotype.
10. detect a method for influenza A H5 and/or H9 hypotype, it is characterized in that, comprise,
Sample to be tested is added to the region being coated with the fluorescently-labeled first antibody of band and the fluorescently-labeled second antibody of band in the first coated film in the arbitrary kit of claim 1-8;
Detect the fluorescence intensity in the first area in described kit, second area and the 3rd region, obtain first area fluorescence intensity, second area fluorescence intensity and the 3rd region fluorescence intensity;
The first predetermined threshold is greater than based on the first ratio, judge to there is influenza A H5 hypotype in described sample to be tested, and/or, the second predetermined threshold is greater than based on the second ratio, judge to there is influenza A H9 hypotype in described sample to be tested, wherein, the first ratio=first area fluorescence intensity/the 3rd region fluorescence intensity, the second ratio=second area fluorescence intensity/the 3rd region fluorescence intensity;
Optionally, described first predetermined threshold and the second predetermined threshold are 0.025.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368907A (en) * | 2008-07-14 | 2009-02-18 | 马义才 | Quantum dot marking test strip quantitative detection system based on CMOS image sensor |
| CN101379397A (en) * | 2006-01-26 | 2009-03-04 | Hx诊断公司 | Monoclonal antibody for H5 subtype fowl-influenza virus hemagglutinin protein or combined active fragment, and uses thereof |
| CN101553732A (en) * | 2006-08-31 | 2009-10-07 | 大阪府 | Rapid diagnosis method specific to avian influenza virus |
| CN101852800A (en) * | 2010-05-18 | 2010-10-06 | 同昕生物技术（北京）有限公司 | Colloidal gold test strip for semi-quantitatively detecting concentration of tacrolimus drug in human whole blood and detection method |
| KR101211593B1 (en) * | 2010-09-06 | 2013-01-07 | 유한회사 바이오노트 | Diagnostic kit for H9 type avian influenza virus using rapid immunochromatography and the method for diagnosing H9 type avian influenza by using the same |
| CN104076142A (en) * | 2014-03-05 | 2014-10-01 | 广东医学院附属医院 | Fluorescent microsphere lateral chromatographic detection strip for multiple joint inspection of trace target substances as well as preparation method and application thereof |
-
2014
- 2014-11-03 CN CN201410614127.2A patent/CN104407135A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101379397A (en) * | 2006-01-26 | 2009-03-04 | Hx诊断公司 | Monoclonal antibody for H5 subtype fowl-influenza virus hemagglutinin protein or combined active fragment, and uses thereof |
| CN101553732A (en) * | 2006-08-31 | 2009-10-07 | 大阪府 | Rapid diagnosis method specific to avian influenza virus |
| CN101368907A (en) * | 2008-07-14 | 2009-02-18 | 马义才 | Quantum dot marking test strip quantitative detection system based on CMOS image sensor |
| CN101852800A (en) * | 2010-05-18 | 2010-10-06 | 同昕生物技术（北京）有限公司 | Colloidal gold test strip for semi-quantitatively detecting concentration of tacrolimus drug in human whole blood and detection method |
| KR101211593B1 (en) * | 2010-09-06 | 2013-01-07 | 유한회사 바이오노트 | Diagnostic kit for H9 type avian influenza virus using rapid immunochromatography and the method for diagnosing H9 type avian influenza by using the same |
| CN104076142A (en) * | 2014-03-05 | 2014-10-01 | 广东医学院附属医院 | Fluorescent microsphere lateral chromatographic detection strip for multiple joint inspection of trace target substances as well as preparation method and application thereof |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105388292A (en) * | 2015-10-19 | 2016-03-09 | 清华大学深圳研究生院 | Reagent kit and method for joint detection of PCT, CRP and IL-6 |
| CN105866440A (en) * | 2016-05-31 | 2016-08-17 | 常州博闻迪医药科技有限公司 | Detection method of glycosylated hemoglobin of blood |
| CN106053803A (en) * | 2016-06-06 | 2016-10-26 | 安邦（厦门）生物科技有限公司 | Multi-connected detection reagent card adopting immunochromatography and used for respiratory pathogens |
| CN107490684A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin collaurum detection method |
| CN107490699A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin fluorescence immunoassay detection method |
| CN106198976A (en) * | 2016-06-30 | 2016-12-07 | 厦门宝太生物科技有限公司 | A kind of for detecting the reagent card of pepsin concn, test kit and purposes |
| CN106053794A (en) * | 2016-06-30 | 2016-10-26 | 厦门宝太生物科技有限公司 | Reagent card for accurately detecting test object, kit and application |
| CN106841605A (en) * | 2017-03-24 | 2017-06-13 | 南通伊仕生物技术股份有限公司 | A kind of thyrotropic hormone Test paper and preparation method thereof |
| CN108318691A (en) * | 2017-04-17 | 2018-07-24 | 清华大学深圳研究生院 | Influenza A and Type B influenza virus lateral chromatography detection kit and method |
| CN108051592A (en) * | 2017-11-28 | 2018-05-18 | 清华大学深圳研究生院 | The detection kit of A type H7 subtype influenza virus and its application |
| CN108107206A (en) * | 2017-11-28 | 2018-06-01 | 清华大学深圳研究生院 | The detection kit of A type H5 subtype influenza virus and its application |
| CN108107207A (en) * | 2017-11-28 | 2018-06-01 | 清华大学深圳研究生院 | The detection kit of A type H9 subtype influenza virus and its application |
| CN108535475A (en) * | 2018-03-28 | 2018-09-14 | 韶关学院 | Ternary system Immune competition method detects the quantum dot immune chromatography detection card and detection method of sulindac |
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